Protein Components of the Microbial Mercury Methylation Pathway ______________________________________________________________ A Dissertation presented to the faculty of the Graduate School at the University of Missouri - Columbia ____________________________________________________ In partial fulfillment of the requirements for the degree Doctor of Philosophy __________________________________________ by Steven D. Smith Dr. Judy D. Wall, Dissertation Supervisor December 2015 The undersigned, appointed by the dean of the Graduate School, have examined the dissertation titled Protein Components of the Microbial Mercury Methylation Pathway Presented by Steven D. Smith, a candidate for the degree of doctor of philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. ____________________________________________ Dr. Judy D. Wall ____________________________________________ Dr. David W. Emerich ____________________________________________ Dr. Thomas P. Quinn ____________________________________________ Dr. Michael J. Calcutt Acknowledgements I would first like to thank my parents and family for their constant support and patience. They have never failed to be there for me. I would like to thank all members of the Wall Lab. At one time or another each one of them has helped me in some way. In particular I would like to thank Barb Giles for her insight into the dynamics of the lab and for her support of me through these years. I am greatly appreciative to Dr. Judy Wall for the opportunity to earn my PhD in her lab. Her constant support and unending confidence in me has been a great source of motivation. It has been an incredible learning experience that I will carry with me and draw from for the rest of my life. Table of Contents Acknowledgements ………………………………………………………………………………………………………… ii List of Figures …………………………………………………………………………………………………………………. v List of Tables …………………………………………………………………………………………………………………… vii Abstract ………………………………………………………………………………………………………………………….. viii Chapter 1 1.1 Mercury sources in the environment ……………………………………………….……………………….. 1 + 1.2 Bioaccumulation of CH3Hg ……………………………………………………………………………………….. 5 1.3 Minamata Convention ………………………………………………………………................................ 9 1.4 Bacterial mer system ………………………………………………………………................................... 9 1.5 Hg-thiol interactions …………………………………………………………………................................. 10 1.6 Hg cycle and microbial transformations ……………………………………………………………………. 11 1.7 Search for the microbial Hg methylation pathway ……………………………………………………. 13 1.8 Studies of Richard Bartha ………………………………………………………………………………………….. 14 1.9 Alternate methylation pathway ………………………………………………………..……………………… 19 1.10 Lead in to this work and ORNL history …………………………………………………………………….. 20 1.11 Sulfate reducing bacteria and D. desulfuricans ND132 ..……………….…………………………. 21 Chapter 2 2.1 Introduction ……………………………………………………………………………………………………………… 25 2.2 Materials and methods …………………………………………………………………………………………….. 29 2.3 Results ………………………………………………………………………………........................................ 48 2.4 Discussion ………………………………………………………………………………………………………………… 56 Chapter 3 3.1 Introduction ………………………………………………………………………………………………………………..59 3.2 Materials and methods ……………………………………………………………………………………………….63 3.3 Results ……………………………………………………………………………………………………….……………….74 3.4 Discussion ……………………………………………………………………………...................................... 80 Chapter 4 4.1 Introduction ……………………………………………………………………………………………….……………….89 4.1.1 metH and other methyltransferases ……………………………………........................ 89 4.1.2 Corrinoid cofactor assembly ……………………………………...……………………………….94 4.2 Materials and methods ……………………………………………………………………………………………….98 4.3 Results ………………………………………………………………………………......................................... 103 4.4 Discussion ………………………………………………………………………….......................................... 108 Chapter 5 5.1 Conclusions and future work ………………………………………………………………………………………113 References ……………………………………………………………………………………………………………………….118 Vita …………………………………………………………………………………………………………………………………..128 List of Figures All figures and table presented are either original, not copyrighted, or used with permission from the copyright holder. Figures and tables taken from the Smith et al. 2015 Applied Environmental Microbiology publication and the Parks et al. 2013 Science publication are used under terms for use by an author or co-author in their dissertation. All other figures are used with permission for inclusion in a dissertation. 1.1 Hg in Wyoming ice core………………………………………………………………………………………….. 2 1.2 2008 US EPA Fish Hg Advisory Map………………………………………….………………………………. 7 1.3 Mercury biogeochemical cycle ……………………………………………………………………………….. 12 1.4 Proposed carbon flow pathway (Bartha, et al.) ..………………………………………..………….. 16 1.5 Optimal enzymatic conditions for potential methyltransferase………….. …………………. 18 1.6 Desulfovibrio desulfurican ND132 electron micrograph ..……………………………………….. 22 2.1 DND132_1056 (HgcA) and CFeSP domain alignments …………………………………………….. 27 2.2 HgcA hydrophobicity plot ……………………………………………………………………………………….. 28 2.3 hgcA and hgcB gene loci in six known mercury methylating bacteria …..…………………. 28 2.4 Anaerobic glove bag for ND132 cell culture…………………………………………………………….. 39 + 2.5 Schematic of MerX CH3Hg detection system………………………………………………………….. 47 2.6 Southern blots to confirm mutants…………………………………………………………………….… 49-50 2.7 RT-PCR analysis of mutants…………………………………………….…………………………..……….. 51-52 2.8 Growth comparison of ND132 and hgcA and hgcB deletions..…………………………………. 53 + 2.9 CH3Hg production by hgcA and hgcB deletions and complements …………………………. 55 2.10 Proposed Hg methylation cycle …………………………………………………………………………….. 57 3.1 Cartoon of proposed HgcA structure.……………………………………………………………………….. 61 3.2 HgcA cap-helix conservation and proposed corrinoid interaction ……………………………. 62 3.3 pMO4661 and hgcAhgcB site directed mutant constructs……………………………………….. 68 3.4 Primer design example for site-directed mutagenesis …………………………………………….. 69 3.5 Approaches for truncations of HgcA ………………………………………………………………………... 71 3.6 Mercury methylation results for HgcA and HgcB point mutants ………………………………. 76 3.7 Western blot for HgcA in ND132 and truncation mutant …………………………………………. 79 3.8 HgcA amino acid sequence showing regions discussed in Chapter 3 ...…………………….. 85 3.9 Dethiobacter alkiliphilus and ND132 HgcB and HgcA alignments………………………….. 87-88 4.1 Genes targeted for deletion biochemically upstream of HgcA…………………………………… 92 4.2 Methylcobalamin structure ……………………………………………………………………………………… 95 4.3 Soluble domain of HgcA interaction with corrinoid…………………………………………………… 96 + 4.4 CH3Hg production by ND132 methyltransferase mutants …... ………………………………. 103 + 4.5 CH3Hg production by the cobT deletion mutant ..…………………………………………………… 105 5.1 HgcA and HgcB affinity tag constructs …………………………………………………………………….. 115 List of Tables 1.1 Missouri 2015 Fish Advisories …………………………………………………………………………………….. 6 2.1 Bacterial strains and plasmids Chapter 2 ……………………………………………………………… 31-32 2.2 Primers Chapter 2……………………………………………………………………………………………...... 33-37 3.1 Bacterial strains Chapter 3 ………………………………………………………………………………………. 64 3.2 Primers and plasmids Chapter 3 …………………………………………………………………………… 65-67 + 3.3 CH3Hg production by HgcA and HgcB point mutants…..…………………………………………… 77 4.1 Primers Chapter 4 ……………………………………………………………………………………….…………98-101 4.2 Methionine auxotrophy data ………………………………………………………………………..……………104 4.3 Hg methylation in selected ND132 deletions mutants……………………………………….……….106 Protein Components of the Microbial Mercury Methylation Pathway Steven D. Smith Dr. Judy D. Wall, Dissertation Supervisor Abstract + Mercury (Hg) in the environment and the resulting methylmercury (CH3Hg ) produced by microorganisms has emerged as a global concern in the last 50 years after a series of high profile, large scale contamination events, increased burning of fossil fuels, and a more recent major escalation of widespread use of Hg in artisanal gold mining. The Hg cycle in the environment is complex and still not completely known. Among the many different forms of Hg present in the environment, Hg, mainly as Hg(II), has + + been known to be converted to the more toxic CH3Hg by microorganisms. CH3Hg accumulates in the + aquatic food chain and humans are eventually exposed to the toxic CH3Hg through their diets. When + exposed to elevated levels of CH3Hg , symptoms include mainly neurological pathologies along with reduced kidney and liver function. However, until recently the biotic mechanism of the conversion of + Hg(II) to CH3Hg was not known. It has now been shown that a pair of genes, hgcA and hcgB, present in a variety of microorganisms, are necessary for bacterial Hg methylation to occur. The structure and functions of the proteins encoded by these genes are still being explored, and here new data are shown for residues in these proteins that suggest key structural elements necessary for the Hg methylation reaction. Also presented are data from other possible proteins in the carbon flow and cobalamin assembly pathways leading up to Hg methylation by HgcA and HgcB. The new work described here is significant in that it not only adds to the expanding base of knowledge of microbial Hg methylation with the identification of hgcA and hgcB, but also shows specific catalytic residues of HgcA and HgcB that can be further studied in organisms that methylate Hg. These data and the mutants designed to facilitate its acquisition can be used in future efforts to identify other